Studies proposed in this competing renewalapplication intend to identify the mechanisms of effect that allow (1) systemic administration of anti-CD3 antibody with or without selected TLR engagement or (2) the generation of, or adoptive transfer of, regulatory T cells (Tregs), to block progression to diabetes in pre- diabetic or recent onset hyperglycemic NOD mice. The hypothesis being studied is that these proposed therapies share a final common pathway ofeffect. Mechanistic studies in this proposal will use cDNA microarray and novel methods of protein expression available in Core B, to compare gene and protein expression patterns seen in treated mice to those seen in normal NOD disease progression when comparedto patterns seen in non-inflamed NOD.810 tissues (NOD Roadmap data from previous U19 contract),to identify the mechanism(s)of the observedtherapeutic effect. Identification, analysis and characterization of gene and protein expression patterns seen following successful therapy of NOD mice should provide important insights into mechanism of effect, identify surrogate markersof effect, and potentially provide additional or alternative targets for therapy. According to our preliminary data obtained under previous U19 funding, two major phenomenon occur following successful therapy of recently hyperglycemic NOD mice; (1) a change in the immune response profile of islet-infiltrating T cells leading to a reorganization of the gene and protein expression patterns and the architecture of the infiltrating T cells, co-incident with inhibition of the autoimmune destruction of beta cells, and (2) pancreatic beta cell regeneration (wound healing) as a consequence of this change. Four Specific Aims have been proposed to test the following hypothesis: administration of endotoxin contaminated anti-CD3 antibody or adoptive cellular therapy, using Tregs or local TGFp production, acting directly on autpreactive lymphocytes present in the islets and draining lymph nodes, or, as we believe, by activating Tregs, either directly, or secondarily blocks islet beta cell destruction and facilitates islet beta cell regeneration as a form of wound healing. These studies have direct impact on one current therapy of recent onset diabetes in man, the use of anti-CD3 antibodies. Despite extremely successful treatment of recent onset hyperglycemic NOD mice with anti-CD3 antibodies, responses seen in the human trials have been less than spectacular. If our preliminary data are correct, that contaminant endotoxin in the anti-CD3 antibodies used in the mouse model was synergistic, the concept of using TLR agonists in synergy with endotoxin free anti-human-CDS antibodies may provide a major therapeutic advance in the treatment of recent onset or pre-diabetic human disease.